Fc variant antibodies and their uses

ABSTRACT

The invention relates to antibodies directed to α2β1 integrin and their uses, including humanized anti-alpha 2 (α2) integrin antibodies and methods of treatment with anti-α2 integrin antibodies. More specifically the present invention relates to humanized anti-α2 integrin antibodies comprising a heavy chain variable region, a light chain variable region, a human light chain constant region and a variant human IgG1 heavy chain constant region which exhibit altered effector function.

This application is a continuation of U.S. patent application Ser. No.13/578,921, filed Aug. 14, 2012, now U.S. Pat. No. 8,975,376, which is a371 National Phase Entry application of International ApplicationPCT/IB2011/000344, filed Feb. 22, 2011, which designated the U.S. andwhich claims the benefit under 35 U.S.C. § 119 of U.S. Application No.61/307,009, filed Feb. 23, 2010. The contents of these applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention generally relates to antibodies directed to α2β1integrin and their uses, including humanized anti-alpha 2 (α2) integrinantibodies and methods of treatment with anti-α2 integrin antibodies.More specifically the present invention relates to humanized anti-α2integrin antibodies comprising a heavy chain variable region, a lightchain variable region, a human light chain constant region and a varianthuman IgG1 heavy chain constant region which exhibit altered effectorfunction.

BACKGROUND OF THE INVENTION

The integrin α2β1 (Very late antigen 2; VLA-2) is expressed on a varietyof cell types including platelets, vascular endothelial cells,epithelial cells, activated monocytes/macrophages, fibroblasts,leukocytes, lymphocytes, activated neutrophils and mast cells. (Hemler,Annu Rev Immunol 8:365:365-400 (1999); Wu and Santoro, Dev. Dyn.206:169-171 (1994); Edelson et. al., Blood. 103(6):2214-20 (2004);Dickeson et al, Cell Adhesion and Communication. 5: 273-281 (1998)). Themost typical ligands for α2β1 include collagen and laminin, both ofwhich are found in extracellular matrix. Typically the I-domain of theα2 integrin binds to collagen in a divalent-cation dependent mannerwhereas the same domain binds to laminin through both divalent-cationdependent and independent mechanisms. (Dickeson et al, Cell Adhesion andCommunication. 5: 273-281 (1998)). The specificity of the α2β1 integrinvaries with cell type and serves as a collagen and/or laminin receptorfor particular cell types, for example α2β1 integrin is known as acollagen receptor for platelets and a laminin receptor for endothelialcells. (Dickeson et al, J Biol. Chem. 272: 7661-7668 (1997))Echovirus-1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I),endorepellin and multiple collectins and the C1q complement protein arealso ligands for α2β1 integrin. (Edelson et al., Blood 107(1): 143-50(2006)) The α2β1 integrin has been implicated in several biological andpathological processes including collagen-induced platelet aggregation,cell migration on collagen, cell-dependent reorganization of collagenfibers as well as collagen-dependent cellular responses that result inincreases in cytokine expression and proliferation, (Gendron, J. Biol.Chem. 278:48633-48643 (2003); Andreasen et al., J. Immunol.171:2804-2811 (2003); Rao et al., J. Immunol. 165(9):4935-40 (2000)),aspects of T-cell, mast cell, and neutrophil function (Chan et. al., J.Immunol. 147:398-404 (1991); Dustin and de Fougerolles, Curr OpinImmunol 13:286-290 (2001), Edelson et. al., Blood. 103(6):2214-20(2004), Werr, et al., Blood 95:1804-1809 (2000), aspects of delayed typehyersensitivity contact hypersensitivity and collagen-induced arthritis(de Fougerolles et. al., J. Clin. Invest. 105:721-720 (2000);Kriegelstein et al., J. Clin. Invest. 110(12):1773-82 (2002)), mammarygland ductal morphogenesis (Keely et. al., J. Cell Sci. 108:595-607(1995); Zutter et al., Am. J. Pathol. 155(3):927-940 (1995)), epidermalwound healing (Pilcher et. al., J. Biol. Chem. 272:181457-54 (1997)),and processes associated with VEGF-induced angiogenesis (Senger et al.,Am. J. Pathol. 160(1):195-204 (2002)).

Integrin/ligand interactions can facilitate leukocyte extravasation intoinflamed tissues (Jackson et al., J. Med. Chem. 40:3359-3368 (1997);Gadek et al., Science 295(5557):1086-9 (2002), Sircar et al., Bioorg.Med. Chem. 10:2051-2066 (2002)), and play a role in downstream eventsfollowing the initial extravasation of leukocytes from the circulationinto tissues in response to inflammatory stimuli, including migration,recruitment and activation of pro-inflammatory cells at the site ofinflammation (Eble J. A., Curr. Phar. Des. 11(7):867-880 (2005)). Someantibodies that block α2β1 integrin were reported to show impact ondelayed hypersensitivity responses and efficacy in a murine model ofrheumatoid arthritis and a model of inflammatory bowel disease(Kriegelstein et al., J. Clin. Invest. 110(12):1773-82 (2002); deFougerolles et. al., J. Clin. Invest. 105:721-720 (2000) and werereported to attenuate endothelial cell proliferation and migration invitro (Senger et al., Am. J. Pathol. 160(1):195-204 (2002), suggestingthat the blocking of α2β1 integrin might prevent/inhibit abnormal orhigher than normal angiogenesis, as observed in various cancers.

It is anticipated that a therapeutic antibody that binds α2β1 integrin,including the α2β1 integrin on platelets, could result in bleedingcomplications. For example, antibodies targeting other plateletreceptors-such as GPIb (Vanhoorelbeke et al., Curr. Drug TargetsCardiovasc. Haematol. Disord. 3(2):125-40 (2003) or GP IIb/IIIa (Schellet al., Ann. Hematol. 81:76-79 (2002), Nieswandt and Watson, Blood102(2):449-461 (2003), Merlini et al., Circulation 109:2203-2206 (2004))have been associated with thrombocytopenia, although the mechanismsbehind this are not well understood. It has been hypothesized thatbinding of an antibody to a platelet receptor can alter its threedimensional structure, and expose normally unexposed epitopes which thenleads to platelet elimination (Merlini et al., Circulation 109:2203-2206(2004). Indeed, the bleeding complications associated with oral doses ofGP IIa/IIIb antagonists have been described as the “dark side” of thisclass of compounds (Bhatt and Topol, Nat. Rev. Drug Discov. 2(1):15-28(2003)). If α2β1 integrin plays an important role in the movement ofleukocytes through inflammatory tissue, it would be desirable to developtherapeutic agents that could target α2β1 for diseases α2β1integrin-associated disorders and/or cellular processes associated withthe disorders, including cancer, inflammatory diseases and autoimmunediseases, if such agents would not activate platelets. Humanizedantibodies capable of targeting α2β1 integrin, such as the α2β1 integrinon leukocytes, which are not associated with adverse bleedingcomplications are described in WO2007/056858. The humanized anti-α2integrin antibodies described therein represent a novel subgroup ofanti-α2 antibodies, which are characterized by an unexpected lack of invivo bleeding complications and/or by a lack of platelet α2β1 integrinactivation. The IgG4 antibodies disclosed in WO2007/056858, however, donot carry effector functions such as ADCC and/or CDC, which are desiredunder certain circumstances, e.g. for the treatment of α2β1integrin-associated disorders such as cancer, where this functionalityleads to increased efficacy of treatment. Thus, it would be desirable todevelop anti-α2β1 integrin antibodies that would exhibit these effectorfunctions to a high degree.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cell-based CDC assay of selected anti-VLA2 antibodyvariants: (1) anti-VLA2 IgG4; (2) anti-VLA2 IgG1; (3) anti-VLA2 IgG2;(4) anti-VLA2 IgG3; (5) anti-VLA2 IgG1133; (6) anti-VLA2 IgG3133; (7)anti-VLA2 IgG1-S324N; (8) anti-VLA2 IgG1-S298A, (9) anti-VLA2IgG1-E269D; (10) anti-VLA2 IgG1-E269D/S298A/S324N; (11) anti-VLA2IgG1-S298A/S324N; (12) IgG1 control; (13) negative control—no antibody.

FIG. 2 shows cell-based ADCC assay of selected anti-VLA2 antibodyvariants at a final concentration of 0.1 μg/ml. (1) anti-VLA2 IgG4; (2)anti-VLA2 IgG1; (3) anti-VLA2 IgG2; (4) anti-VLA2 IgG3; (5) anti-VLA2IgG1133; (6) anti-VLA2 IgG3133; (7) anti-VLA2 IgG1-S324N; (8) anti-VLA2IgG1-S298A, (9) anti-VLA2 IgG1-E269D; (10) anti-VLA2IgG1-E269D/S298A/S324N; (11) anti-VLA2 IgG1-S298A/S324N; (12) IgG1control; (13) negative control—no antibody.

FIG. 3 shows cell-based ADCC assay of selected anti-VLA2 antibodyvariants at a final concentration of 0.01 μg/ml. (1) anti-VLA2 IgG4; (2)anti-VLA2 IgG1; (3) anti-VLA2 IgG2; (4) anti-VLA2 IgG3; (5) anti-VLA2IgG1133; (6) anti-VLA2 IgG3133; (7) anti-VLA2 IgG1-S324N; (8) anti-VLA2IgG1-S298A, (9) anti-VLA2 IgG1-E269D; (10) anti-VLA2IgG1-E269D/S298A/S324N; (11) anti-VLA2 IgG1-S298A/S324N; (12) IgG1control; (13) negative control—no antibody.

SUMMARY OF THE INVENTION

The present invention provides a humanized anti-α2 integrin antibodycomprising a heavy chain variable region, a light chain variable region,a human light chain constant region and a variant human IgG1 heavyconstant domain, whereas the variant human IgG1 heavy chain constantregion comprises at least one amino acid modification relative to thehuman IgG1 heavy chain constant region of the parent humanized anti-α2integrin antibody, and whereas the antibody exhibits altered effectorfunction compared to the parent humanized anti-α2 integrin antibody.

The invention further provides an isolated nucleic acid encoding theabove-mentioned humanized anti-α2β1 integrin antibody.

The invention further provides a vector comprising the above-mentionednucleic acid.

The invention further provides a host cell comprising theabove-mentioned nucleic acid or the above-mentioned vector.

The invention further provides a composition comprising theabove-mentioned humanized anti-α2 integrin antibody and apharmaceutically acceptable carrier.

The invention further provides a kit comprising the above-mentionedhumanized anti-α2 integrin or the above-mentioned composition andinstructions for the treatment of an α2β1 integrin-associated disorder.

The invention further provides a method of treating an α2β1integrin-associated disorder in a subject, the method comprisingadministering to the subject a therapeutically effective amount of theabove-mentioned anti-α2 integrin antibody or the above-mentionedcomposition.

The invention further provides a method for inhibiting leukocyte bindingto collagen comprising administering to a subject an amount of theabove-mentioned anti-α2β1 integrin antibody or the above-mentionedcomposition effective to inhibit the binding of the leukocytes tocollagen.

The invention further provides a use of the above mentioned humanizedanti-α2 integrin antibody or the above-mentioned composition as amedicament.

The invention further provides a use of the above mentioned humanizedanti-α2 integrin antibody or the above-mentioned composition for thetreatment of an α2β1 integrin-associated disorder.

The invention further provides the above mentioned humanized anti-α2integrin antibody or the above mentioned composition for use in a methodfor the treatment of an α2β1 integrin-associated disorder.

The invention further provides a use of the above mentioned humanizedanti-α2 integrin antibody or the above-mentioned composition for thepreparation of a medicament for the treatment of an α2β1integrin-associated disorder.

The invention further provides an antibody comprising a variant humanIgG Fc region which comprises amino acid substitution S324N replacingserine at amino acid position 324 of the parent antibody withasparagine, whereas the antibody exhibits improved complement dependentcytotoxicity (CDC) and improved antibody dependent cell mediatedcytotoxicity (ADCC) as compared to the parent antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to humanized anti-α2 integrin antibodieswhich exhibit altered effector function compared to the parent humanizedanti-α2 integrin antibody.

α2β1 integrin is a molecule comprised of an α2 integrin subunit from thefamily of alpha integrins, and a β1 integrin subunit from the family ofbeta integrins, and may be from any subject including a mammal, butpreferably is from a human. The α2β1 integrin may be purified from anynatural source, or may be produced synthetically (e.g., by use ofrecombinant DNA technology). The nucleic acid coding sequences for α2integrin and for β1 integrin are described in Takada and Hemler J. CellBiol. 109(1):397-407 (1989; GenBank submission X17033; subsequentlyupdated to entry NM 002203) and Argraves, W. S, J. Cell. Biol. September105(3):1183-90 (1987; Genbank submission X07979.1 and related sequencesrepresenting alternatively spliced variants), respectively.

The ‘I’ domain of the α2β1 integrin molecule refers to a region of thisα2β1 integrin molecule within the α2 subunit, and is described, forexample, in Kamata et al., J Biol. Chem. 269:9659-9663(1994); Emsley etal., J. Biol. Chem. 272:28512 (1997) and Cell 101:47 (2000). The Idomain of α2 integrin contains a MIDAS type of ligand binding site(Metal Ion Dependent Adhesion Site) which has a requirement and aspecificity for a given divalent cation to support ligand binding.

An α2 integrin-associated disorder includes a disorder, disease, orcondition that involves. α2 integrin-dependent processes/function (e.g.,binding, activity) that mediate aberrant cellular reactions withintarget tissue. An α2 integrin-associated disorder refers to a disorder,disease, symptom or condition that involves α2 integrin-dependentprocesses/function (e.g., binding, activity) that mediate aberrantcellular reactions within target tissue. Examples of α2integrin-dependent processes involved in disease includecollagen-dependent cellular responses such as those involved inincreases in cytokine expression and proliferation, aspects of T-cell-,mast cell- and neutrophil-function, inflammatory disorders, mammarygland ductal morphogenesis, epidermal wound healing, and angiogenesis.Examples of α2 integrin-associated disorders include, but are notlimited to, inflammatory diseases or disorders including but not limitedto inflammatory bowel disease (such as Crohn's disease and ulcerativecolitis), reactions to transplant (including transplant rejection),optic neuritis, spinal cord trauma, rheumatoid arthritis, multiplesclerosis (including treatment of neurological sequelae associatedtherewith as well as multiple sclerosis characterized by relapse),autoimmune diseases or disorders (including systemic lupus erythematosus(SLE), diabetes mellitus, Reynaud's syndrome, experimental autoimmuneencephalomyelitis, Sjorgen's syndrome, scleroderma), juvenile onsetdiabetes, and disorders associated with abnormal or higher than normalangiogenesis (such as diabetic retinopathy, age related maculadegeneration, cardiovascular disease, psoriasis, rheumatoid arthritisand cancer) as well as infections that induce an inflammatory response.

Treatment of an α2β1 integrin-associated disorder includes boththerapeutic use and prophylactic or preventative use of the anti-α2integrin antibodies described herein. Those in need of treatment includethose already diagnosed with the disorder as well as those in which theonset of the disorder is to be prevented or delayed.

The terms “anti-α2 integrin antibodies” or “antibody that bind to α2” or“antibody that bind to α2 integrin subunit” or “anti-VLA-2 antibodies”are used synonymously herein and include antibodies, preferablyhumanized IgG antibodies, that bind to human α2 integrin, e.g. that bindto immobilized α2β1 with an affinity (Kd) of 50 nM or less, preferably10 nM or less, more preferably 1 nM or less, in particular 0.5 nM orless.

By the terms “parent humanized anti-α2 integrin antibody” or “parentanti-α2 integrin antibody” or “parent anti-VLA-2 antibody” which areused synonymously herein are meant an antibody that binds human-α2integrin, e.g. a humanized IgG1 anti-α2 integrin antibody which can bemodified to comprise a variant human IgG1 heavy chain constant region.The parent humanized anti-α2 integrin antibody is identical to theantibody which comprises a variant human IgG1 heavy chain constantregion, except for the amino acid modification in the human IgG1 heavychain constant region and is usually an antibody with a native humanIgG1 heavy chain constant region. The amino acid modification ispreferably not isotopic.

The term “antibody” or “immunoglobulin” is used in the broadest sense,and covers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, and multispecific antibodies, solong as they exhibit the desired biological activity. The term antibodyor immunoglobulin comprises full length antibodies as well as fragmentsthereof which have antigen binding properties, i.e. which bind to α2integrin. The term “antibody” includes a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding fragment thereof. Each heavychain is comprised of a heavy chain variable region (abbreviated hereinas VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CHI, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region (abbreviated herein as CL). The lightchain constant region is comprised of one domain. The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR or FW). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the First component (C1q)of the classical complement system.

The term “full length antibody” as used herein includes the structurethat constitutes the natural biological form of an antibody, includingvariable and constant regions. For example, in most mammals, includinghumans and mice, the full length antibody of the IgG class is a tetramerand consists of two identical pairs of two immunoglobulin chains, eachpair having one light and one heavy chain, each light chain comprisingimmunoglobulin domains VL and CL, and each heavy chain comprisingimmunoglobulin domains VH, CH1 (C[gamma]1), CH2 (C[gamma]2), and CH3(C[gamma]3). In some mammals, for example in camels and llamas, IgGantibodies may consist of only two heavy chains, each heavy chaincomprising a variable domain attached to the Fc region.

The term “chimeric antibody” as used herein includes antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The term “humanized antibody” as used herein includes antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences as well as within the CDR sequencesderived from the germline of another mammalian species.

The term “human antibody” as used herein includes antibodies havingvariable regions, in which both the framework and CDR regions arederived from human germline immunoglobulin sequences. Furthermore, ifthe antibody contains a constant region, the constant region also isderived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g. mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo). However, the term “human antibody”, as used herein, is notintended to include antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences.

A monoclonal antibody includes an antibody obtained from a population ofsubstantially homogeneous antibodies, e.g., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site. Furthermore, in contrast to conventional (e.g.,polyclonal) antibody preparations which typically include differentantibodies directed against different determinants (e.g., epitopes) onan antigen, each monoclonal antibody is directed against at least asingle determinant on the antigen. The modifier “monoclonal” indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, monoclonal antibodies may be made by the hybridoma method firstdescribed by Kohler et al., Nature 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonalantibodies may also be isolated from phage antibody libraries, forexample, using the techniques described in Clackson et al., Nature352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).Monoclonal antibodies can also be isolated using the techniquesdescribed in U.S. Pat. Nos. 6,025,155 and 6,077,677 as well as U.S.Patent Application Publication Nos. 2002/0160970 and 2003/0083293 (seealso, e.g., Lindenbaum, et al., Nucleic Acids Research 32 (21):0177(2004)).

A hypervariable region includes the amino acid residues of an antibodywhich are responsible for antigen-binding. The hypervariable regioncomprises amino acid residues from a complementarity determining regionor CDR (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in thelight chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3)in the heavy chain variable domain; Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)) and/or those residues from ahypervariable loop (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3)in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101(H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol.196: 901-917 (1987)). Framework or FR residues are those variable domainresidues other than the hypervariable region residues. For antibodiesdescribed herein, the CDR and framework regions are identified based onthe Kabat numbering system except that the CDR1 of the heavy chain isdefined by Oxford Molecular's AbM definition as spanning residues 26 to35. The Oxford Molecular's AbM antibody modeling software(http://people.cryst.cck.ac.uk/˜ubc07s/) (Martin et al., Proc. NatlAcad. Sci. USA, 86, 9268-9272 (1989); Martin et al., Methods Enzymol.,203, 121-153 (1991); Pedersen et al., Immunomethods, 1, 126 (1992); andRees et al., In Sternberg M. J. E. (ed.), Protein Structure Prediction.Oxford University Press, Oxford, 141-172. (1996)) combines the Kabat CDRand the Chothia hypervariable region numbering systems to define CDRs.

The term “amino acid modification” herein includes an amino acidsubstitution, insertion, and/or deletion in a polypeptide sequence. By“amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with another amino acid. For example, thesubstitution R94K refers to a variant polypeptide, in this case a heavychain variable framework region variant, in which the arginine atposition 94 is replaced with a lysine. For the preceding example, 94Kindicates the substitution of position 94 with a lysine. For thepurposes herein, multiple substitutions are typically separated by aslash. For example, R94K/L78V refers to a double variant comprising thesubstitutions R94K and L78V. By “amino acid insertion” or “insertion” asused herein is meant the addition of an amino acid at a particularposition in a parent polypeptide sequence. For example, insert −94designates an insertion at position 94. By “amino acid deletion” or“deletion” as used herein is meant the removal of an amino acid at aparticular position in a parent polypeptide sequence. For example, R94-designates the deletion of arginine at position 94.

For all immunoglobulin heavy chain constant region positions discussedin the present invention, numbering is according to the EU index as inKabat (Kabat et al., 1991, Sequences of Proteins of ImmunologicalInterest, 5th Ed., United States Public Health Service, NationalInstitutes of Health, Bethesda, incorporated entirely by reference). Thenumbering of the immunoglobulin heavy chain constant region positions isreferred herein as “numbering system set forth in Kabat” or “EU index asin Kabat” which is equivalently used herein and designates numberingaccording to the EU index as in Kabat. The “EU index as in Kabat” refersto the residue numbering of the human IgGI EU antibody, as described inEdelman et al., 1969, PNAS 63:78-85.

Antibodies are grouped into classes, also referred to as isotypes, asdetermined genetically by the constant region. Human constant lightchains are classified as kappa (CK) and lambda (C[lambda]) light chains.Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. The IgG class is the most commonly used for therapeuticpurposes. In humans this class comprises subclasses IgG1, IgG2, IgG3,and IgG4. In mice this class comprises subclasses IgG1, IgG2a, IgG2b,IgG3. IgM has subclasses, including, but not limited to, IgM1 and IgM2.IgA has several subclasses, including but not limited to IgA1 and IgA2.Thus, “isotype” as used herein is meant any of the classes or subclassesof immunoglobulins defined by the chemical and antigenic characteristicsof their constant regions. The known human immunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

The term “Fc” or “Fc region”, as used herein includes the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (C[gamma]2 and C[gamma]3) and the hinge betweenCgammaI (C[gamma]I) and Cgamma2 (C[gamma]2). Although the boundaries ofthe Fc region may vary, the human IgG heavy chain Fc region is usuallydefined to comprise residues C226 or P230 to its carboxyl-terminus,wherein the numbering is according to the EU index as in Kabat. Fc mayrefer to this region in isolation, or this region in the context of anFc polypeptide, for example an antibody.

By “variant human IgG1 heavy chain constant region” as used herein ismeant a human IgG1 heavy chain constant region that differs from aparent human IgG1 heavy chain constant region by virtue of at least oneamino acid modification. By “Fc variant” or “variant Fc” or “varianthuman IgG1 Fc region” as used herein is meant an Fc sequence thatdiffers from that of a parent Fc sequence by virtue of at least oneamino acid modification. A variant human IgG1 heavy chain constantregion or an Fc variant comprises one or more amino acid modificationsrelative to a parent Fc polypeptide, wherein said amino acidmodification(s) provide one or more optimized properties. A varianthuman IgG1 heavy chain constant region or a Fc variant of the presentinvention differs in amino acid sequence from its parent IgG1 by virtueof at least one amino acid modification. Thus variant human IgG1 heavychain constant region or Fc variants of the present invention have atleast one amino acid modification compared to the parent. Alternatively,the variant human IgG1 heavy chain constant region of the presentinvention may have more than one amino acid modification as compared tothe parent, e.g. may comprise conversion of a whole constant regionimmunoglobulin domain or, preferably, of an Fc region of one isotype ina different isotype, e.g. the conversion of the Fc region of the humanIgG1 heavy chain constant region to an Fc region from human IgG3resulting in an isotypic variant comprising the CHI from human IgG1, thehinge from human IgG1 and the Fc region from human IgG3. Modificationsmay be made genetically using molecular biology, or may be madeenzymatically or chemically.

The Fc variants of the present invention may be substantially encoded byany allotype or isoallotype of any immunoglobulin gene. In a preferredembodiment, the Fc variants of the present invention find use inantibodies or Fc fusions that comprise IgG1 sequences that areclassified as G1m(1), G1m(2), G1m(3), G1m(17), nG1m(1), nG1 m(2), and/ornG1 m(17). Thus in the context of an IgGI isotype, the Fc variants ofthe present invention may comprise a Lys (G1m(17)) or Arg (G1m(3)) atposition 214, an Asp356/Leu358 (G1m(1)) or Glu356/Met358 (nG1 m(1)),and/or a Gly (G1m(2)) or Ala (nG1m(2)) at position 431.

The term “isotypic variant” as used herein includes an amino acidmodification that converts at least one amino acid of one isotype,preferably at least one amino acid of the heavy chain constant region ofone isotype, to the corresponding amino acid in a different, alignedisotype. The amino acid modification may comprise conversion of a wholeconstant region immunoglobulin domain or, preferably; of an Fc region ofone isotype in a different isotype, e.g. the conversion of the Fc regionof the human IgG1 heavy chain constant region to an Fc region from humanIgG3 resulting in an isotypic variant comprising the CHI from humanIgG1, the hinge from human IgG1 and the Fc region from human IgG3.

By “hinge” or “hinge region” or “antibody hinge region” herein is meantthe flexible polypeptide comprising the amino acids between the firstand second constant domains of an antibody. Structurally, the IgG CH1domain ends at EU position 220, and the IgG CH2 domain begins at residueEU position 237. Thus for IgG the antibody hinge is herein defined toinclude positions 221 (D221 in IgGI) to 231 (A231 in IgGI), wherein thenumbering is according to the EU index as in Kabat.

The term “effector function” as used herein includes a biochemical eventthat results from the interaction of an antibody Fc region with an Fcreceptor or ligand. The term “effector function” as used herein includesphagocytosis, opsonization, cell binding, resetting, complementdependent cytotoxicity (CDC), C1q binding, binding affinity of theantibody for an Fc[gamma] receptor or antibody dependent cell mediatedcytotoxicity (ADCC). Preferably the effector function is complementdependent cytotoxicity (CDC) and/or antibody dependent cell mediatedcytotoxicity (ADCC). The effector function is measured by standard invitro assays, which are known in the art and commercially available.Usually ADCC is measured by the lactate dehydrogenase (LDH)-releasingassay as described in Example 2 of the present application and CDC ismeasure by the cell-based assay described in Example 1 of the presentapplication.

The term “alter effector function” or “exhibiting altered effectorfunction” as used herein includes exhibition of enhanced effectorfunction of an antibody, e.g. a humanized anti-α2 integrin antibody,comprising a variant human IgG1 heavy chain constant region compared tothe parent antibody, i.e. the effector function of the antibodycomprising a variant human IgG1 heavy chain constant region is more than10%, preferably more than 20%, more preferably more than 30%, mostpreferably more than 50%, in particular more than 60%, most particularmore than 70% higher than the effector function of the parent antibody.

The term “ADCC” or “antibody dependent cell-mediated cytotoxicity” asused herein includes the cell-mediated reaction wherein nonspecificcytotoxic cells that express Fc[gamma]Rs recognize bound antibody on atarget cell and subsequently cause lysis of the target cell. In variousaspects, the enhanced ADCC effector function can mean enhanced potencyor enhanced efficacy. By “potency” as used in the experimental contextis meant the concentration of antibody when a particular therapeuticeffect is observed EC50 (half maximal effective concentration). By“efficacy” as used in the experimental context is meant the maximalpossible effector function at saturating levels of antibody.

The term “CDC” or “complement dependent cytotoxicity” as used hereinincludes the reaction wherein one or more complement protein componentsrecognize bound antibody on a target cell and subsequently cause lysisof the target cell.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc. Preferably thesubject is human.

A cytotoxic agent includes a substance that inhibits or prevents thefunction of cells and/or causes destruction of cells. The can includeradioactive isotopes (e.g., ¹³¹I, ¹²⁵I, ⁹⁰Y and ¹⁸⁶Re), chemotherapeuticagents, and toxins such as enzymatically active toxins of bacterial,fungal, plant or animal origin, or fragments thereof. A non-cytotoxicagent refers to a substance that does not inhibit or prevent function ofcells and/or does not cause destruction of cells. A non-cytotoxic agentmay include an agent that can be activated to become cytotoxic. Anon-cytotoxic agent may include a bead, liposome, matrix or particle(see, e.g., U.S. Patent Publications 2003/0028071 and 2003/0032995 whichare incorporated by reference herein). Such agents may be conjugated,coupled, linked or associated with an anti-α2β1 integrin antibody asdescribed herein.

A chemotherapeutic agent refers to a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include but arenot limited to Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosinearabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Taxotere (docetaxel),Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan,Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan and other relatednitrogen mustards.

An isolated nucleic acid molecule refers to a nucleic acid molecule thatis identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the source e.g. inthe natural source of the antibody nucleic acid. An isolated nucleicacid molecule is other than in the form or setting in which it is foundin nature. Isolated nucleic acid molecules therefore are distinguishedfrom the nucleic acid molecule as it exists in natural cells. However,an isolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the antibody where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations include progeny. Transformants and transformed cells(e.g., obtained by transfection, transformation or transduction ofnucleic acids, vectors, virus, etc.) include the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny that have the same function or biological activity asscreened for in the originally transformed cell are included. Wheredistinct designations are intended, it will be clear from the context.

Humanized Anti-α2 Integrin Antibodies Comprising a Variant Human IgG1Heavy Constant Domain

The present invention provides a humanized anti-α2 integrin antibodycomprising a heavy chain variable region, a light chain variable region,a human light chain constant region and a variant human IgG1 heavy chainconstant region, whereas the variant human IgG1 heavy chain constantregion comprises at least one amino acid modification relative to thehuman IgG1 heavy constant region of the parent humanized anti-α2integrin antibody, and whereas the antibody exhibits altered effectorfunction compared to the parent humanized anti-α2 integrin antibody.

In one aspect, the present disclosure provides a humanized anti-α2integrin antibody comprising a heavy chain variable region, a lightchain variable region, a human light chain constant region and a varianthuman IgG1 heavy constant region, wherein the variant human IgG1 heavychain constant region is an isotypic variant comprising the CH1 fromhuman IgG1, the hinge from human IgG1 and the Fc region from human IgG3.

In one embodiment the isotypic variant human IgG1 heavy constant regioncomprises SEQ ID NO: 35.

In one aspect, the present disclosure provides a humanized anti-α2integrin antibody comprising a heavy chain variable region, a lightchain variable region, a human light chain constant region and a varianthuman IgG1 heavy constant region, wherein the variant human IgG1 heavychain constant region is an isotypic variant comprising the CH1 fromhuman IgG3, the hinge from human IgG1 and the Fc region from human IgG3.

In one embodiment the isotypic variant human IgG1 heavy constant regioncomprises SEQ ID NO: 36.

In one aspect, the present disclosure provides a humanized anti-α2integrin antibody comprising a heavy chain variable region, a lightchain variable region, a human light chain constant region and a varianthuman IgG1 heavy constant region, wherein the variant human IgG1 heavychain constant region comprises a variant human IgG1 Fc region whichcomprises at least one amino acid modification relative to the human IgGFc region of the parent humanized anti-α2 integrin antibody.

In one embodiment, the amino acid modification comprises an amino acidsubstitution at amino acid position selected from the group consistingof 269, 298, and 324, preferably an amino acid substitution at aminoacid position 298 and/or 324, wherein the amino acid position of eachgroup member is indicated utilizing the numbering system set forth inKabat.

In another embodiment, the amino acid modification comprises an aminoacid substitution selected from the group consisting of E269D, S298A,and S324N, preferably amino acid substitutions S298A and/or S324N,wherein the amino acid position of each group member is indicatedutilizing the numbering system set forth in Kabat.

In another embodiment, the amino acid modification comprises acombination of amino acid substitutions at amino acid position selectedfrom the group consisting of 269/298, 269/324, 298/324, and 269/298/324,preferably 298/324, or 269/298/324, wherein the amino acid position ofeach group member is indicated utilizing the numbering system set forthin Kabat.

In another embodiment, the amino acid modification comprises acombination of amino acid substitutions selected from the groupconsisting of E269D/S298A, E269D/S324N, S298A/S324N, andE269D/S298A/S324N, preferably S298A/S324N or E269D/S298A/S324N, whereinthe amino acid position of each group member is indicated utilizing thenumbering system set forth in Kabat.

In one embodiment the variant human IgG1 Fc region comprises a sequenceselected from the group consisting of SEQ ID NOs: 37-43.

The effector function altered is usually complement dependentcytotoxicity (CDC) and/or C1q binding and/or antibody dependent cellmediated cytotoxicity (ADCC) and/or binding affinity of the antibody foran Fc[gamma] receptor, preferably complement dependent cytotoxicity(CDC) and/or antibody dependent cell mediated cytotoxicity (ADCC). CDC,C1q binding, ADCC, and binding affinity of the antibody for an Fc[gamma]receptor are measured by standard in vitro assays, which are known inthe art and commercially available. Usually ADCC is measured by thelactate dehydrogenase (LDH)-releasing assay as described e.g. in Example2 of the present application and CDC is measure by the cell-based assaydescribed e.g. in Example 1 of the present application.

Preferably the humanized anti-α2 integrin antibody of the presentdisclosure comprising the variant human IgG1 heavy chain constant regionexhibits improved CDC in an in vitro assay as described above comparedto the parent humanized antibody. “Exhibition of improved CDC” or“exhibiting improved CDC” as used herein includes a) exhibition ofenhanced CDC compared to the parent antibody, i.e. the parent humanizedanti-α2 integrin antibody already exhibits CDC which is enhanced by theamino acid modification of the human IgG1 heavy chain constant regionand b) de novo exhibition of CDC compared to the parent humanizedanti-α2 integrin antibody, i.e. the parent humanized anti-α2 integrinantibody does not exhibit CDC, thus CDC has been introduced de novo bythe amino acid modification of the human IgG1 heavy chain constantregion.

Thus in a further aspect the present disclosure provides a humanizedanti-α2 integrin antibody comprising a heavy chain variable region, alight chain variable region, a human light chain constant region and avariant human IgG1 heavy constant region, whereas the variant human IgG1heavy chain constant region comprises at least one amino acidmodification relative to the human IgG1 heavy chain constant region ofthe parent humanized anti-α2 integrin antibody, and whereas the antibodyexhibits improved complement dependent cytotoxicity (CDC) as compared tothe parent humanized antibody. A preferred variant human IgG1 heavychain constant region of the humanized anti-α2 integrin antibody whichexhibits improved complement dependent cytotoxicity (CDC) as compared tothe parent humanized antibody comprises a variant human IgG1 Fc regionwhich comprises an amino acid substitution selected from the groupconsisting of S324N, S298A/S324N, and E269D/S298A/S324N, more preferablythe variant human IgG1 Fc region comprises the amino acid sequenceselected from the group consisting of SEQ ID NO: 39, 42 and 43.

In one embodiment, the humanized anti-α2 integrin antibody comprisingthe variant human IgG1 heavy chain constant region which exhibitsimproved complement dependent cytotoxicity (CDC) as compared to theparent humanized anti-α2 integrin antibody exhibits antibody dependentcell mediated cytotoxicity (ADCC) equivalent to the parent humanizedantibody. Exhibition of ADCC equivalent to the parent humanized antibodyincludes an ADCC of ±50%, preferably ±40%, more preferably ±30%, mostpreferably ±20%, in particular ±10%, of the ADCC of the parent humanizedantibody. It is known that IgG1 antibodies, e.g humanized IgG1 anti-α2integrin antibodies as parent antibodies exhibit ADCC. It is notpredictable, however, if a modification, e.g. a substitution of an aminoacid which provides for improved complement dependent cytotoxicity (CDC)of the IgG1 heavy chain constant region does have an impact on ADCC.Thus the humanized anti-α2 integrin antibodies of the present inventionwhich comprises a variant human IgG1 heavy chain constant region whichexhibits improved CDC surprisingly exhibits ADCC equivalent to theparent humanized antibody.

In a further aspect the present disclosure provides a humanized anti-α2integrin antibody comprising a heavy chain variable region, a lightchain variable region, a human light chain constant region and a varianthuman IgG1 heavy constant region, wherein the variant human IgG1 heavychain constant region comprises a variant human IgG1 Fc region whichcomprises at least one amino acid modification relative to the human IgGFc region of the parent humanized anti-α2 integrin antibody, and whereasthe antibody exhibits improved antibody dependent cell mediatedcytotoxicity (ADCC) as compared to the parent humanized antibody. Apreferred variant human IgG1 heavy chain constant region of thehumanized anti-α2 integrin antibody which exhibits improved antibodydependent cell mediated cytotoxicity (ADCC) as compared to the parenthumanized antibody comprises a variant human IgG1 Fc region whichcomprises an amino acid substitution selected from the group consistingof E269D, S298A, S298A/S324N, and E269D/S298A/S324N, more preferably thevariant human IgG1 Fc region comprises the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 37, 38, 42 and 43.

In a further aspect the present disclosure provides a humanized anti-α2integrin antibody comprising a heavy chain variable region, a lightchain variable region, a human light chain constant region and a varianthuman IgG1 heavy constant region, wherein the variant human IgG1 heavychain constant region comprises a variant human IgG1 Fc region whichcomprises at least one amino acid modification relative to the human IgGFc region of the parent humanized anti-α2 integrin antibody, wherein theamino acid modification is amino acid substitution S298A/S324N orE269D/S298A/S324N, whereas the antibody exhibits improved complementdependent cytotoxicity (CDC) and improved antibody dependent cellmediated cytotoxicity (ADCC) as compared to the parent humanized anti-α2integrin antibody.

Antibodies of the present invention have been constructed comprisingCDRs from both the heavy chain variable and light chain variable regionsof the murine monoclonal antibody clone BHA2.1 (Hangan et al., CancerRes. 56:3142-3149 (1996)). Preferred starting materials for constructingantibodies are anti-α2 integrin antibodies such as those secreted by theBHA2.1 hybridoma (e.g., TMC-2206) that are function-blocking antibodiesdirected against human α2 integrin and are dependent for binding andactivity on the presence of an intact I-domain within the targeted α2integrin. Preferred are humanized antibodies with the epitopespecificity of TMC-2206 (or BHA2.1), including antibodies which bind tothe inactive conformation of the α2 integrin molecule, and/or do not actas ligand mimetics. Preferred are humanized antibodies with the epitopespecificity of TMC-2206 (or BHA2.1) that, although they interact withα2β1 integrin present on both leukocytes and platelets, do not causeplatelet activation, impair aggregation of activated platelets oncollagen, have minimal or no effect on bleeding and/or are notassociated with bleeding complications at administered concentrations,including therapeutic doses in vivo.

Thus also provided is the above-mentioned humanized anti-α2 integrinantibody comprising a heavy chain variable region comprising HCDR1comprising the amino acid sequence GFSLTNYGIH (SEQ ID NO:1), HCDR2comprising the amino acid sequence VIWARGFTNYNSALMS (SEQ ID NO:2) andHCDR3 comprising the amino acid sequence ANDGVYYAMDY (SEQ ID NO:3).

Also provided is the above-mentioned humanized anti-α2 integrin antibodycomprising a light chain variable region comprising LCDR1 comprising theamino acid sequence SAQSSVNYIH (SEQ ID NO:4), LCDR2 comprising the aminoacid sequence DTSKLAS (SEQ ID NO:5) and LCDR3 comprising the amino acidsequence QQWTTNPLT (SEQ ID NO:6).

Also provided is the above-mentioned humanized anti-α2 integrin antibodycomprising a heavy chain variable region comprising HCDR1 comprising theamino acid sequence GFSLTNYGIH (SEQ ID NO:1), HCDR2 comprising the aminoacid sequence VIWARGFTNYNSALMS (SEQ ID NO:2) and HCDR3 comprising theamino acid sequence ANDGVYYAMDY (SEQ ID NO:3); and/or a light chainvariable region comprising LCDR1 comprising the amino acid sequenceSAQSSVNYIH (SEQ ID NO:4), LCDR2 comprising the amino acid sequenceDTSKLAS (SEQ ID NO:5) and LCDR3 comprising the amino acid sequenceQQWTTNPLT (SEQ ID NO:6).

In an embodiment, the above-mentioned heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 7.

In an embodiment, the above-mentioned heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 7 in which (a) position71 is Lys, (b) position 73 is Asn, (c) position 78 is Val, or (d) anycombination of (a)-(c).

In an embodiment, the above-mentioned heavy chain variable regioncomprises an amino acid sequence selected from SEQ ID NOs:8-19.

In an embodiment, the above-mentioned heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 17.

In an embodiment, the above-mentioned heavy chain variable regionfurther comprises a FW4 region comprising the amino acid sequenceWGQGTLVTVSS (SEQ ID NO:20).

In an embodiment, the above-mentioned light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 21.

In an embodiment, the above-mentioned light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 21 in which (a) position2 is Phe, (b) position 45 is Lys, (c) position 48 is Tyr, or (d) anycombination of (a)-(c).

In an embodiment, the above-mentioned the light chain variable regioncomprises an amino acid sequence selected from SEQ ID NOs: 22-33.

In an embodiment, the above-mentioned light chain variable regioncomprises the amino acid sequence of SEQ. ID NO: 30.

In an embodiment, the above-mentioned light chain variable regionfurther comprises a FW4 region comprising the amino acids sequenceFGQGTKVEIK (SEQ ID NO: 34).

Further provided is the above-mentioned humanized anti-α2 integrinantibody comprising the above-mentioned variant human IgG1 heavy chainconstant region, the above-mentioned heavy and light chain variableregions and a human light chain constant region.

Thus in a further embodiment, the anti-α2 integrin antibody comprises aheavy chain comprising SEQ ID NO: 47 and a light chain comprising SEQ IDNO: 56.

In a further embodiment, the anti-α2 integrin antibody comprises a heavychain comprising SEQ ID NO: 48 and a light chain comprising SEQ ID NO:56.

In a further embodiment, the anti-α2 integrin antibody comprises a heavychain selected from the group consisting of SEQ ID NO: 49-55 and a lightchain comprising SEQ ID NO: 56.

Further provided is an antibody comprising a variant human IgG Fc regionwhich comprises amino acid substitution S324N replacing serine at aminoacid position 324 of the parent antibody with asparagine, whereas theantibody exhibits improved complement dependent cytotoxicity (CDC).

Further provided is an antibody comprising a variant human IgG Fc regionwhich comprises amino acid substitution S324N replacing serine at aminoacid position 324 of the parent antibody with asparagine, whereas theantibody exhibits improved complement dependent cytotoxicity (CDC) andimproved antibody dependent cell mediated cytotoxicity (ADCC) ascompared to the parent antibody. The antibody may further comprise aminoacid substitution E269D replacing glutamate at amino acid position 269of the parent antibody with aspartate and/or substitution S298Areplacing serine at amino acid position 298 of the parent antibody withalanine. The antibody can be selected from the group consisting of achimeric antibody, a humanized antibody and a fully human antibody Theantibody is preferably a humanized antibody, more preferably a humanizedanti-α2 integrin antibody.

In an embodiment, the above-mentioned humanized anti-α2 integrinantibody recognizes the I domain of human α2 integrin.

In an embodiment, the above-mentioned humanized anti-α2 integrinantibody binds α2β1 integrin.

In an embodiment, the above-mentioned humanized anti-α2 integrinantibody inhibits binding of α2 or α2β1 integrin to an α2β1 integrinligand. Usually the α2β1 integrin ligand is selected from collagen,laminin, Echovirus-1, decorin, E-cadherin, matrix metalloproteinase I(MMP-I), endorepellin, collectin and C1q complement protein and ispreferably collagen.

Also provided is an isolated nucleic acid encoding the above-mentionedhumanized anti-α2 integrin antibody, a vector comprising the nucleicacid and a host cell comprising the nucleic acid or the vector.

Also provided is a composition comprising the above-mentioned humanizedanti-α2 integrin antibody and a pharmaceutically acceptable carrier.

Also provided is a method of treating an α2β1 integrin-associateddisorder in a subject, the method comprising administering to thesubject a therapeutically effective amount of the above-mentionedhumanized anti-α2 integrin antibody or the above-mentioned composition.The α2β1 integrin-associated disorder includes inflammatory disease,autoimmune disease and a disease characterized by abnormal or increasedangiogenesis, in particular inflammatory bowel disease, Crohn's disease,ulcerative colitis, reactions to transplant, optical neuritis, spinalcord trauma, rheumatoid arthritis, systemic lupus erythematosus (SLE),diabetes mellitus, multiple sclerosis, Reynaud's syndrome, experimentalautoimmune encephalomyelitis, Sjorgen's syndrome, scleroderma, juvenileonset diabetes, diabetic retinopathy, age related macular degeneration,cardiovascular disease, psoriasis, cancer as well as infections thatinduce an inflammatory response more particular multiple sclerosis,rheumatoid arthritis, optical neuritis and spinal cord trauma.

Cancers which can be treated by the above-mentioned humanized anti-α2integrin antibody or the above-mentioned composition are selected fromthe group consisting of squamous cell cancer, lung cancer includingsmall-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, and squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom'sMacroglobulinemia; chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema such as that associated with brain tumors, Meigs' syndrome,melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma,and epidermoid carcinoma. Cancers which are preferably treated using theanti-α2 integrin antibodies described herein are selected from the groupconsisting of breast cancer, colorectal cancer, rectal cancer, non-smallcell lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer,prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma,kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma,ovarian cancer, mesothelioma, and multiple myeloma. The cancerousconditions amendible for treatment of the invention include metastaticcancers. Thus even more preferred are cancers selected from the groupconsisting of breast cancer, colorectal cancer, rectal cancer, non-smallcell lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer,prostate cancer, metastatic prostate cancer, liver cancer, pancreaticcancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, headand neck cancer, melanoma, ovarian cancer, mesothelioma, multiplemyeloma, metastatic colorectal and metastatic breast cancer. Particularpreferred are cancers selected from the group consisting of non-smallcell lung cancer, pancreatic cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, breast cancer, colon cancer, colorectalcancer, kidney cancer, prostate cancer, metastatic prostate cancer,mesothelioma, fibrosarcoma, osteosarcoma, epidermoid carcinoma,metastatic colorectal, metastatic prostate and metastatic breast cancer.More particular preferred are cancers selected from the group consistingof non-small cell lung cancer, pancreatic cancer, glioblastoma, livercancer, breast cancer, colon cancer, colorectal cancer, kidney cancer,prostate cancer, mesothelioma, fibrosarcoma, metastatic colorectal,metastatic prostate and metastatic breast cancer. Even more particularpreferred are cancers selected from the group consisting of pancreaticcancer, breast cancer, colon cancer, colorectal cancer, non-small celllung cancer, fibrosarcoma, metastatic colorectal, prostate cancer,metastatic prostate cancer and metastatic breast cancer. Most particularpreferred are cancers selected from the group consisting of pancreaticcancer, breast cancer, colon cancer, colorectal cancer, non-small celllung cancer, and fibrosarcoma. Most preferred are pancreatic cancer,breast cancer or metastatic breast cancer, with a particular preferenceto pancreatic cancer. Equally most particular preferred is prostatecancer or metastatic prostate cancer. “Breast cancer” as referred hereininclude mammary adenocarcinoma. The method of the present invention isparticularly suitable for the treatment of vascularized tumors.

Preferably the method is not associated with (a) platelet activation,(b) platelet aggregation, (c) a decrease in circulating platelet count,(d) bleeding complications, or (e) any combination of (a) to (d).

Also provided is a method for inhibiting leukocyte binding to collagencomprising administering to a subject an amount of the above-mentionedhumanized anti-α2 integrin antibody effective to inhibit the binding ofthe leukocytes to collagen.

Also provided is a kit comprising the above-mentioned humanized anti-α2integrin antibody or the above-mentioned composition according andinstructions for the treatment of an α2β1 integrin-associated disorder.

Construction of Humanized Anti-α2 Integrin Antibodies and Conjugates

Antibodies may be constructed wherein the human acceptor molecule forthe light chain variable region is selected based on homologyconsiderations between potential acceptor molecule variable regions andwith the light chain variable region of the murine antibody. Germlinecandidate human acceptor molecules are preferred to reduce potentialimmunogenicity. Germline databases are made up of antibody sequencesthat read through the end of the heavy chain FW3 region and partiallyinto the CDR3 sequence. For selection of a FW4 region, it is preferredto search databases of mature antibody sequences which have been derivedfrom the selected germline molecule, and also preferred to select areasonably homologous FW4 region for use in the recombinant antibodymolecule. Human acceptor molecules are preferably selected from the samelight chain class as the murine donor molecule, and of the samecanonical structural class of the variable region of the murine donormolecule. Secondary considerations for selection of the human acceptormolecule for the light chain variable region include homology in CDRlength between the murine donor molecule and the human acceptormolecule. Human acceptor antibody molecules are preferably selected byhomology searches to the V-BASE database, and other databases such asthe Kabat and the public NCBI databases may be used as well. Forhumanized anti-α2 integrin antibodies with the same or similar epitopespecificity and/or functional properties as TMC-2206, a preferred lightchain human acceptor molecule is the germline antibody sequence A14 forthe FW 1-3 region and the sequence FGQGTKVEIK for FW4 (SEQ ID NO:34)which represents a common FW-4 of mature kappa 1 light chains (e.g.,light chain sequence AAB24132 (NCBI entry gi/259596/gb/AAB24132).

Antibodies may be constructed wherein the human acceptor molecule forthe heavy chain variable region is selected based on homologyconsiderations between potential acceptor molecule variable regions andthe heavy chain variable region of the murine antibody. Germlinecandidate human acceptor molecules are preferred to reduce potentialantigenicity. Germline databases are made up of antibody sequences thatread through the end of the heavy chain FW3 region and partially intothe CDR3 sequence. For selection of a FW4 region, it is preferred tosearch databases of mature antibody sequences which have been derivedfrom the selected germline molecule, and also preferred to select areasonably homologous FW4 region for use in the recombinant antibodymolecule. Human acceptor molecules are preferably selected from the sameheavy chain class as the murine donor molecule, and of the samecanonical structural class of the variable region of the murine donormolecule. Secondary considerations for selection of the human acceptormolecule for the heavy chain variable region include homology in CDRlength between the murine donor molecule and the human acceptormolecule. Human acceptor antibody molecules are preferably selected byhomology search to the V-BASE database, although other databases such asthe Kabat and the public NCBI databases may be used as well. For anti-α2integrin antibodies with the same or similar epitope specificity and/orfunctional properties as TMC-2206, a preferred heavy chain acceptormolecule is the germline antibody sequence 4-59 for the FW 1-3 regionand antibody, CAA48104.1 (NCBI entry, gi/33583/emb/CAA48104.1) a matureantibody derived from the 4-59 germline sequence for the FW 4 region(SEQ ID NO:20).

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (e.g., U.S. Pat. No. 6,204,023). Monoclonalantibodies may also be made using the techniques described in U.S. Pat.Nos. 6,025,155 and 6,077,677 as well as U.S. Patent ApplicationPublication Nos. 2002/0160970 and 2003/0083293 (see also, e.g.,Lindenbaum, et al., Nucleic Acids Research 32 (21):0177 (2004)).

Amino acid sequence variants of humanized anti-α2β1 integrin antibodyare prepared by introducing appropriate nucleotide changes into ahumanized anti-α2β1 integrin antibody DNA, or by peptide synthesis. Suchvariants include, for example, deletions from, and/or insertions intoand/or substitutions of, residues within the amino acid sequences shownfor the anti-α2 integrin antibody of the present invention. Anycombination of amino acid deletion, insertion, and substitution is madeto arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid changes also mayalter post-translational processes of the humanized anti-α2 integrinantibody, such as changing the number or position of glycosylationsites.

There are a number of methods used to make antibodies human orhuman-like (e.g., “humanization”). Approaches to humanize antibodieshave varied over the years. One approach was to generate murine variableregions fused to human constant regions, so-called murine-human Fcchimeras (see, e.g., Morrison et al, Proc. Natl. Acad. Sci. USA81:6851-6855 (1984); U.S. Pat. No. 5,807,715). Another approachexploited the fact that CDRs could be readily identified based on theirhypervariable nature (Kabat et al, J. Biol. Chem. 252:6609-6616 (1977)),Kabat, Adv. Protein Chem. 32:1-75 (1978)) and canonical structure(Chothia and Lesk, J. Mol. Biol. 196(4):901-17 (1987); Lazakani et al.,J. Mol. Biol. 272:929 (1997) and humanized by grafting just thenon-human CDR regions (referred to as donor CDRs) onto a human framework(referred to as acceptor frameworks) as shown, for example by Jones etal., Nature 321(6069):522-5 (1986); (see, e.g., U.S. Pat. Nos.5,225,539; 6,548,640). The six CDR loops are presented in a cluster, andbased on crystallographic analysis, critical framework residues withinthe so-called “Vernier” zone flanking the CDRs or in the heavy-lightchain interface can be readily identified (see, e.g., Chothia and Lesk,J. Mol. Biol. 196(4):901-17 (1987); Chothia et al., J. Mol. Biol.186(3):651-63 (1985); Chothia et al., Nature 342(6252):877-83 (1989)).These residues can be back-mutated to the murine residue to restore thecorrect relative orientation of the six CDRs (see, e.g., Verhoyen etal., Science 239(4847):1534-6 (1988); Reichman et al., Nature332(6162):323-7 (1988); Tempest et al., Biotechnology (NY) 9(3):266-71(1991)). Since variable regions can be classified in families that bearrelatively high homology between mouse and human (reviewed in e.g.,Pascual and Capra Adv. Immunol. 49:1-74 (1991)), these early studiesalso indicated that the potential for loss in affinity could beminimized in the grafted antibody by selecting the human germlinesequence with the highest homology to the murine antibody of interestfor use as the human acceptor molecule (see, e.g., U.S. Pat. No.5,225,539; Verhoyen et al., Science 239(4847):1534-6 (1988)).

Methods for humanizing a non-human α2 integrin antibody are describede.g. in WO2007/056858. In order to humanize an anti-α2 integrinantibody, the nonhuman antibody starting material is obtained, includingby preparation from immunization or by purchase of commerciallyavailable antibodies. Exemplary techniques for humanizing antibodiesused in the present invention e.g for humanizing TMC-2206 are describedin WO2007/056858.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2)neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic:asn, gln, his, lys, arg; (5) residues that influence chain orientation:gly, pro; and (6) aromatic: trp, tyr, phe. Any cysteine residue notinvolved in maintaining the proper confirmation of a humanized anti-α2integrin antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody and/or adding one ormore glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatit contains or lacks one or more of the above-described tripeptidesequences (for N-linked glycosylation sites). The alteration may also bemade by the addition of, substitution by, or deletion of, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites). Nucleic acid molecules encodingamino acid sequence variants of humanized anti-α2 integrin antibody areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring amino acid sequence variants) or preparationby oligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, or cassette mutagenesis of an earlier prepared variant or anon-variant version of humanized anti-α2 integrin antibody.

Ordinarily, amino acid sequence variants of a humanized anti-α2 integrinantibody, will have an amino acid sequence having at least 75% aminoacid sequence identity with the original humanized antibody amino acidsequences of either the heavy or the light chain (e.g., variable regionsequences as in SEQ ID NO:17 or SEQ ID NO:30, respectively), morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, and most preferably at least 95%, including for example,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, and 100%. Identity or homology withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical with thehumanized anti-α2 integrin residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions (asdescribed above) as part of the sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theantibody sequence shall be construed as affecting sequence identity orhomology. Thus sequence identity can be determined by standard methodsthat are commonly used to compare the similarity in position of theamino acids of two polypeptides. Using a computer program such as BLASTor FASTA, two polypeptides are aligned for optimal matching of theirrespective amino acids (either along the full length of one or bothsequences, or along a pre-determined portion of one or both sequences).The programs provide a default opening penalty and a default gappenalty, and a scoring matrix such as PAM250 (a standard scoring matrix;see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5,supp. 3 (1978)) can be used in conjunction with the computer program.For example, the percent identity can the be calculated as: the totalnumber of identical matches multiplied by 100 and then divided by thesum of the length of the longer sequence within the matched span and thenumber of gaps introduced into the longer sequences in order to alignthe two sequences.

In some embodiments, it may be desirable to generate multispecific(e.g., bispecific) humanized anti-α2 integrin antibodies having bindingspecificities for at least two different epitopes. Exemplary bispecificantibodies (e.g., with two different binding arms) may bind to twodifferent epitopes of the α2β1 integrin protein. Alternately, an anti-α2integrin arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2 orCD3), or Fc receptors for IgG (FcγR), such as FcγR1 (CD64), FcγRII(CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms ona cell which has α2β1 integrin bound to its surface. Bispecificantibodies can be used to localized cytotoxic agents to cells with α2β1integrin bound to their surface. These antibodies possess a α2β1integrin binding arm and an arm which binds the cytotoxic agent (e.g.,gelonin, saporin, anti-interferon alpha, vinca alkaloid, ricin A chain,or radioisotope hapten).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain of an antibody constant domain. In this method,one or more small amino acid side chains are replaced with larger sidechains (e.g., tyrosine or tryptophan). Compensatory cavities ofidentical or smaller size to the large side chain(s) are created on theinterface of the second antibody by replacing large amino acid sidechains with smaller ones (e.g., alanine or threonine). This provides amechanism for increasing the yield of the heterodimers over otherunwanted end-products such as homodimers (see, e.g., WO96/27011).

Bispecific antibodies include cross-linked or heteroconjugateantibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed, for example, in U.S. Pat. No. 4,676,980 along with a numberof cross-linking techniques.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared (see, e.g., Tutt et al., J.Immunol. 147:60 (1991)).

Immunoconjugates comprising a humanized anti-α2 integrin antibodyconjugated to a moiety, e.g., a molecule, composition, complex, oragent, for example a cytotoxic agent such as a chemotherapeutic agent,toxin (e.g., an enzymatically active toxin of bacterial, fungal, plantor animal origin, or fragments thereof), or a radioactive isotope (e.g.,a radioconjugate), for the targeting of the agent to an anti-α2integrin-expressing cell, tissue or organ. Such an immunoconjugate maybe used in a method of targeting the moiety or agent to a particularsite of action characterized by the presence of α2 or α2β1 integrin.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin or the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-alpha 2 integrin antibodies. Examples include²¹²Bi, ¹³¹In, ⁹⁰Y or ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as gluteraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), or bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionuclide to the antibody (see, e.g., WO94/11026).

In another embodiment, the antibody may be conjugated to a receptor(such as streptavidin) for utilization in pretargeting α2integrin-expressing cell, tissue or organ wherein the antibody-receptorconjugate is administered to the patient, followed by removal of unboundconjugate from the circulation using a clearing agent and thenadministration of a ligand (e.g., avidin) which is conjugated to anagent, for example a cytotoxic agent (e.g., a radio-nuclide).

The anti-α2 integrin antibodies disclosed herein may also be formulatedas immunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Humanized anti-α2 integrin antibodies may also be used in AntibodyDirected Enzyme Prodrug Therapy (ADEPT) by conjugating the antibody to aprodrug-activating enzyme which converts a prodrug (e.g., a peptidylchemotherapeutic agent, see, e.g., WO81/01145) to an active drug. (see,e.g., WO88/07378 and U.S. Pat. No. 4,975,278).

Enzymes may be covalently bound to the anti-α2 integrin antibodies bytechniques well known in the art, including the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of ananti-α2 integrin antibody linked to at least a functionally activeportion of an enzyme can be constructed using recombinant DNA techniqueswell known in the art (see, e.g., Neuberger et al., Nature 312: 604-608(1984)).

Covalent modifications of the humanized anti-α2 integrin antibodies maybe made, for example, by chemical synthesis or by enzymatic or chemicalcleavage of the antibody. Other types of covalent modifications of theantibody are introduced into the molecule by reacting targeted aminoacid residues of the antibody with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues. Cysteinyl residues, for example, most commonly are reactedwith α-haloacetates (and corresponding amines), such as chloroaceticacid or chloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues, for example, are derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0. Lysinyl and amino-terminal residues, for example, are reactedwith succinic or other carboxylic acid anhydrides. Derivatization withthese agents, has the effect of reversing the charge of the lysinylresidues. Other suitable reagents for deriyatizing α-amino-containingresidues include imidoesters such as methyl picolinimidate, pyridoxalphosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate. Arginyl residues, for example, are modified by reactionwith one or several conventional reagents, among them phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginine residues requires that the reaction be performed in alkalineconditions because of the high pK_(a) of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group. Tyrosyl residues, for example, arespecifically modified with particular interest in introducing spectrallabels into tyrosyl residues by reaction with aromatic diazoniumcompounds or tetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form P-acetyl tyrosyl species and 3-nitroderivatives, respectively. Tyrosyl residues are iodinated using ¹²⁵I or¹³¹I to prepare labeled proteins for use in radioimmunoassay. Carboxylside groups, for example, aspartyl or glutamyl, are selectively modifiedby reaction with carbodiimides (R—N═C═N—R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions. Glutaminyl andasparaginyl residues are frequently deamidated to the correspondingglutamyl and aspartyl residues, respectively. These residues aredeamidated under neutral or basic conditions. The deamidated form ofthese residues falls within the scope of this invention. Othermodifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine (see, e.g., WO87/05330;Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981)).

Removal of any carbohydrate moieties present on the antibody may beaccomplished, for example, chemically or enzymatically. Chemicaldeglycosylation requires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact (see, e.g., Hakimuddin, et al., Arch. Biochem. Biophys.259: 52 (1987); Edge et al., Anal. Biochem., 118: 131 (1981)). Enzymaticcleavage of carbohydrate moieties on antibodies can be achieved by theuse of a variety of endo- and exo-glycosidases, (see, e.g., Thotakura etal., Meth. Enzymol. 138: 350 (1987)).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, such aspolyethylene glycol, polypropylene glycol, or polyoxyalkylenes (see,e.g., U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337).

Isolated nucleic acid(s) encoding a humanized anti-α2 integrin antibody,as well as vectors and host cells comprising the nucleic acid, andrecombinant techniques for the production of the antibody are describedherein. For recombinant production of the antibody, the nucleic acid(s)encoding the antibody are isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

An anti-α2 integrin antibody may be produced recombinantly, including asa fusion polypeptide with a heterologous polypeptide, which ispreferably a signal sequence or other polypeptide having a specificcleavage site at the N-terminus of the mature protein or polypeptide.The heterologous signal sequence selected preferably is one that isrecognized and processed (e.g., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processa eukaryotic signal sequence (e.g., an immunoglobulin signal sequence),the signal sequence is substituted by a prokaryotic signal sequenceincluding, for example, pectate lysase (such as pelB), alkalinephosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders.For yeast secretion, a yeast signal sequence may be utilized, including,for example, the yeast invertase leader, a factor leader (includingSaccharomyces and Kluyveromyces α-factor leaders), or acid phosphataseleader, the C. albicans glucoamylase leader, or the signal described inWO90/13646. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available and may be utilized. The DNA for such a precursorregion (e.g., the signal sequence) is ligated in reading frame to DNAencoding an anti-α2 integrin antibody.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors, this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. For example, the origin of replication from the plasmid pBR322is suitable for most gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (e.g., the SV40 origin may typically beused only because it contains the early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, (e.g., the gene encoding D-alanine racemase for Bacilli).

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs methotrexate, neomycin, histidinol, puromycin, mycophenolicacid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theanti-α2 integrin antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-α2 integrin antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker, including an aminoglycosidic antibiotic, such askanamycin, neomycin, or G418 (see e.g., U.S. Pat. No. 4,965,199).

One suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1 (see, e.g., Jones, Genetics, 85: 12 (1977)). The presence ofthe trp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6μ circular plasmid pKD1 can beused for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis by Van den Berg, Bio/Technology,8:135 (1990). Stable multi-copy expression vectors for secretion ofmature recombinant human serum albumin by industrial strains ofKluyveromyces have also been disclosed (see, e.g., Fleer et al.,Bio/Technology, 9: 968-975 (1991)).

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the anti-α2integrin antibody nucleic acid. Promoters suitable for use withprokaryotic hosts include the arabinose promoter (e.g., araB), phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgamo (S.D.) sequence operably linked to the DNA encoding theanti-α2 integrin antibody.

Promoter sequences are known for eukaryotes. Most eukaryotic genes havean AT-rich region located approximately 25 to 30 bases upstream from thesite where transcription is, initiated. Another sequence found 70 to 80bases upstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. Such sequences aresuitably inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts includebut are not limited to the promoters for 3-phosphoglycerate kinase orother glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other yeast promoters, which are inducible promoters havingthe additional advantage of transcription controlled by growthconditions, are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, metallothionein, glyceraldehyde-3-phosphatedehydrogenase, and enzymes responsible for maltose and galactoseutilization. Suitable vectors and promoters for use in yeast expressionare further described in EP 73,657. Yeast enhancers also areadvantageously used with yeast promoters.

Anti-α2 integrin antibody transcription from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus or Simian Virus 40(SV40), from heterologous mammalian promoters, for example, the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment that also contains the SV40 viral origin ofreplication. The immediate early promoter of the human cytomegalovirusis conveniently obtained as a HindIII E restriction fragment. A systemfor expressing DNA in mammalian hosts using the bovine papilloma virusas a vector is disclosed in U.S. Pat. No. 4,419,446, and a modificationof this system is described in U.S. Pat. No. 4,601,978 (see, also Reyeset al., Nature 297: 598-601 (1982) on expression of human β-interferoncDNA in mouse cells under the control of a thymidine kinase promoterfrom herpes simplex virus). Alternatively, the rous sarcoma virus longterminal repeat can be used as the promoter.

Transcription of DNA encoding an anti-α2 integrin antibody by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Often, however,an enhancer from a eukaryotic cell virus is used. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers (see,also, e.g., Yaniv, Nature 297: 17-18 (1982) on enhancing elements foractivation of eukaryotic promoters). The enhancer may be spliced intothe vector at a position 5′ or 3′ to the anti-α2 integrinantibody-encoding sequence, but is preferably located at a site 5′ fromthe promoter. Other gene regulation systems well known in the art (e.g.inducible systems, such as tetracycline inducible systems andGeneSwitch™) can be used to control the transcription of DNA encoding ananti-α2 integrin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding an anti-α2 integrin antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region (see, e.g., WO94/11026 and the expression vectordisclosed therein).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells as describedabove. Suitable prokaryotes for this purpose include eubacteria,including gram-negative or gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis, Pseudomonas such as P.aeruginosa, and Streptomyces. Suitable E. coli cloning hosts include E.coli 294 (ATCC 31,446), E. coli B, E. coli X1776 (ATCC 31,537), and E.coli W3110 (ATCC 27,325).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-alpha 2integrin antibody-encoding vectors. Saccharomyces cerevisiae, or commonbaker's yeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and Strainsare commonly available and useful, such as Schizosaccharomyces pombe;Kluyveromyces hosts including K. lactis, K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, or K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi includingNeurospora, Penicillium, Tolypocladium, or Aspergillus hosts such as A.nidulans or A. niger.

Suitable host cells for the expression of glycosylated anti-α2 integrinantibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, for example, the L-1variant of Autographa californica NPV and the Bm-5 strain of Bombyx moriNPV, and such viruses may be used, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells, including a variety of mammalian cells, has becomeroutine procedure. Examples of useful mammalian host cells include: amonkey kidney CV1 line transformed by SV40 (e.g., COS-7, ATCC CRL 1651);a human embryonic kidney line 293 or 293 cells subcloned for growth insuspension culture (see e.g., Graham et al., J. Gen Virol. 36: 59(1977)); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinesehamster ovary (CHO) cells, including CHO cells lacking DHFR (see, e.g.,DHFR Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mousesertoli cells ((e.g., TM4, Mather, Biol. Reprod. 23: 243-251 (1980));monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidneycells (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells(e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34);buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells(e.g., W138, ATCC CCL 75); human liver cells (e.g., Hep G2, HB 8065);mouse mammary tumor (e.g., MMT 060562, ATCC CCL51); TRI cells (see,e.g., Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5cells; FS4 cells; or a human hepatoma line (e.g., Hep G2).

Host cells are transformed with an above-described expression or cloningvectors for anti-α2 integrin antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants and/or amplifying the genes encodingthe desired sequences.

The host cells used to produce an anti-α2 integrin antibody may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58: 44 (1979), Barnes at al., Anal.Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S.patent Re. No. 30,985 may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Cultureconditions, such as temperature, pH, and the like, are selected by thoseskilled in the art, including those culture conditions previously usedwith the host cell selected for expression.

Anti-α2 integrin antibodies can be purified from cells, includingmicrobial or mammalian cells using, for example, protein Achromatography, ion exchange chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and/or affinitychromatography. The suitability of protein A as an affinity liganddepends on the species and isotype of any immunoglobulin Fc domain thatis present in the antibody. Protein A can be used to purify antibodiesthat are based on human γ1, γ2, or γ4 heavy chains (see, e.g., Lindmarket al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is useful for mouseisotypes and for human γ3 (see, e.g., Guss et al, EMBO J. 5:1516-1517(1986)). The matrix to which the affinity ligand is attached is mostoften agarose, but other matrices are available. Mechanically stablematrices such as controlled pore glass or poly(styrenedivinyl)benzeneallow for faster flow rates and shorter processing times than can beachieved with agarose. Where the antibody comprises a CH3 domain, theBakerbond ABX™ (J. T. Baker, Phillipsburg, N.J.) is useful forpurification. Protein purification can include one or more of thefollowing techniques such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™, chromatography on an anion orcation exchange resin (e.g., a polyaspartic acid column),chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and/orhydrophobic interaction chromatography. For example, it may be usefulfollowing any purification step(s), to subject a mixture comprising theantibody of interest and contaminants to low pH hydrophobic interactionchromatography using an elution buffer at a pH between about 2.5-4.5,preferably performed at low salt concentrations (e.g., from about0-0.25M salt).

Formulations of an anti-α2 integrin antibody, including those fortherapeutic administration, are prepared for storage by mixing theantibody having the desired degree of purity with optionalphysiologically acceptable carriers, diluents, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, diluents, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, or other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Fortherapeutic uses the anti-α2 integrin antibody of the present inventionmay be formulated e.g. in phosphate buffered saline (PBS) containing0.03% Tween-80™.

The antibody formulation may also contain more than one active compoundfor the particular indication being treated, preferably those withcomplementary activities that do not adversely affect each other. It maybe desirable to use anti-α2 integrin antibody in addition to one or moreagents currently used to prevent or treat the disorder in question. Inaddition, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles ornanocapsules) or in macroemulsions. Such techniques are disclosed, forexample, in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

Formulations to be used for in vivo administration are preferablysterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Therapeutic Uses of Humanized Anti-α2 Integrin Antibodies

An anti-α2 integrin antibody may be used to treat various α2β1 integrinassociated disorders as described herein. The anti-α2 integrin antibodyis administered by any suitable means, including parenteral,subcutaneous, intraperitoneal, intrapulmonary, or intranasal. If desiredfor local immunosuppressive treatment, intralesional administration ofthe antibody (including perfusing or otherwise contacting the graft withthe antibody before transplantation) is done. Parenteral administrationincludes intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the anti-α2 integrin antibodyis suitably administered by pulse infusion, for example, with decliningdoses of the antibody. Preferably the dosing is given by injections,most preferably intravenous or subcutaneous injections. This may dependin part on whether the administration is brief or chronic. Morepreferably the anti-α2 integrin antibodies or the compositions asdescribed herein are administered in the methods of the presentinvention by intravenous infusion, intravenous bolus, subcutaneousadministration, subcutaneous infusion or subcutaneous bolus, whereasintravenous infusion or intravenous bolus is most preferred. The term“intravenous infusion” refers to introduction of a drug into the vein ofan animal or human patient over a period of time greater thanapproximately 5 minutes, preferably between approximately 30 to 90minutes, although, according to the invention, intravenous infusion isalternatively administered for 10 hours or less. The term “intravenousbolus” or “intravenous push” refers to drug administration into a veinof an animal or human such that the body receives the drug inapproximately 15 minutes or less, preferably 5 minutes or less. The term“subcutaneous administration” refers to introduction of a drug under theskin of an animal or human patient, preferable within a pocket betweenthe skin and underlying tissue, by relatively slow, sustained deliveryfrom a drug receptacle. The pocket may be created by pinching or drawingthe skin up and away from underlying tissue. The term “subcutaneousinfusion” refers to introduction of a drug under the skin of an animalor human patient, preferably within a pocket between the skin andunderlying tissue, by relatively slow, sustained delivery from a drugreceptacle for a period of time including, but not limited to, 30minutes or less, or 90 minutes or less. The term “subcutaneous bolus”refers to drug administration beneath the skin of an animal or humanpatient, where bolus drug delivery is preferably less than approximately15 minutes, more preferably less than 5 minutes, and most preferablyless than 60 seconds. Administration is preferably within a pocketbetween the skin and underlying tissue; where the pocket is created, forexample, —by pinching or drawing the skin up and away from underlyingtissue. Optionally, the infusion may be made by subcutaneousimplantation of a drug delivery pump implanted under the skin of theanimal or human patient, wherein the pump delivers a predeterminedamount of drug for a predetermined period of time, such as 30 minutes,90 minutes, or a time period spanning the length of the treatmentregimen. Intermittent or periodic dosing is a dosing that is continuousfor a certain period of time and is at regular intervals that arepreferably separated more than by one day.

“Therapeutically effective amount” or “effective amount” which are usedsynonymously herein, refer to an amount of the anti-α2 integrinantibodies described herein effective to ameliorate or prevent thesymptoms, or prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure provided herein. The term “therapeutically effectiveamount” of the anti-α2 integrin antibodies described herein specificallyrefers to the amount needed to delay or inhibit cancer e.g. tumorgrowth.

For the prevention or treatment of an α2β1 integrin-associated disorder,the appropriate dosage of antibody will depend on the type of disease tobe treated, as defined above, the severity and course of the disease,whether the anti-α2 integrin antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments.

The anti-α2 integrin antibodies can be thus administered to a subject,preferably to human, in the method of the present invention, at atherapeutically effective amount ranging from about 0.1. to about 100mg/kg. Preferably, a therapeutically effective amount ranging from about1 to about 20 mg/kg, more preferably a therapeutically effective amountranging from about 3 to about 10 mg/kg is administered to a subject,preferably to human. A therapeutically effective amount of the humanizedantibody or binding fragment thereof can be administered to the subjectin one or more therapeutically effective doses.

For the prevention or treatment of an α2β1 integrin-associated disorder,the appropriate dosage of antibody will depend on the type of disease tobe treated, as defined above, the severity and course of the disease,whether the anti-α2 integrin antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments.

Depending on the type and severity of an α2β1 integrin-associateddisorder from about 0.1 mg/kg to about 100 mg/kg of antibody is aninitial candidate dosage for administration to the subject, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage to e.g. human might range from 0.1 mg/kto 20 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be usefule.g. a once every two weeks dosis regimen seems preferable. The progressof this therapy is readily monitored by those skilled in the art.

An anti-α2 integrin antibody composition will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, results from pharmacological and toxicity studies andother factors known to medical practitioners. A therapeuticallyeffective amount of the antibody to be administered is determined byconsideration of such, and is the minimum amount necessary to prevent,ameliorate, or treat an α2β1 integrin-associated disorder. Such amountis preferably below the amount that is toxic to the host or renders thehost significantly more susceptible to infections.

The anti-α2 integrin antibody need not be, but may be optionallyformulated, co-administered or used as an adjunct therapy with one ormore agents currently used to prevent or treat the disorder in question.For example, in rheumatoid arthritis, the antibody may be given inconjunction with a glucocorticosteroid, Remicaid® or any approvedtreatment for rheumatoid arthritis. For multiple sclerosis, the antibodymay be given in conjunction with an interferonβ, Avonex, Copaxon, orother approved therapies for treatment of the signs and symptoms ofmultiple sclerosis. For transplants, the antibody may be administeredconcurrently with or separate from an immunosuppressive agent as definedabove, such as cyclosporin A, to modulate the immunosuppressant effect.Alternatively, or in addition, α2β1 integrin antagonists may beadministered to the mammal suffering from an α2β1 integrin-associateddisorder. The effective amount of such other agents depends on theamount of anti-α2 integrin antibody present in the formulation, the typeof disorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLES Example 1: Humanized Anti-Alpha2 Integrin Antibody Variantswith Enhanced Complement-Mediated Effector Function

The humanized anti-α2 integrin antibodies described therein represent anovel subgroup of anti-α2 (anti-VLA2) antibodies, which arecharacterized by an unexpected lack of in vivo bleeding complicationsand/or by a lack of platelet α2β1 integrin activation. The IgG4antibodies disclosed in WO2007/056858, however, do not carry effectorfunctions such as ADCC and/or CDC, which are desired under certaincircumstances, e.g. for the treatment of α2β1 integrin-associatedcancers, where this functionality can lead to increased efficacy oftreatment. Thus, it would be desirable to develop anti-α2β1 integrinantibodies that would exhibit these effector functions to a high degree.

The four isotypes of human IgG differ from each other in the potenciesof effector functions and other activities. In general, the rank orderof potency is IgG1≥IgG3>>IgG4≥IgG2 for ADCC and IgG3≥IgG1>>IgG2=IgG4 forCDC (Niwa R. et al., J Immunol Methods 2005; 306:151-60). In this study,one anti-VLA-2 IgG4 antibody disclosed in WO2007/056858 (with heavychain SEQ ID 57, and light chain SEQ ID NO: 56) was used to create a newsubgroup of anti-VLA2 antibodies consisting of naturally occurring humanantibody isotype variants IgG1, IgG2, and IgG3; engineered human IgG1isotype variants, and chimeras of human IgG1 and human IgG3 isotypevariants. All variants were created by mutagenesis techniques based onoverlap PCR assembly methods.

To generate full antibody variants, each newly created heavy chainvector was transfected with the same anti-VLA-2 kappa light chainvector, carrying the anti-VLA-2 kappa light chain cDNA coding lightchain sequence (SEQ ID NO: 56) and having identical expressionregulating elements as the heavy chain vectors described below.Consequently, the heavy chain of a novel anti-VLA2 antibody variantdefines a novel full antibody variant; therefore a heavy chain and thecorresponding full antibody are designated by the same name.

Variants Based on Naturally Occurring Human Antibody Isotypes

To create naturally occurring isotype variants heavy chains, the cDNA ofthe anti-VLA-2 IgG4 antibody encoding the heavy chain hinge and constantdomains of SEQ ID NO: 57 (Kabat residue 119 (corresponding to residue121 in SEQ ID NO: 57) to its C-terminus) were replaced by thecorresponding cDNA sequence encoding amino-acid for the hinge andconstant domains of human IgG1 (NCBI GenBank accession no. J00228.1,sequence from residue 1 to its C-terminus), human IgG2 (NCBI GenBankaccession no. J00230.1, sequence from residue 1 to its C-terminus), andhuman IgG3 (NCBI GenBank accession no. X03604.1, sequence from residue 2to its C-terminus). These naturally occurring heavy chain variantssubsequently define novel anti-VLA2 antibodies which are designatedheavy chain anti-VLA2 IgG1 (SEQ ID NO: 44), heavy chain anti-VLA2 IgG2(SEQ ID NO: 45), and heavy chain anti-VLA2 IgG3 (SEQ ID NO: 46)respectively.

Variants Based on Human IgG1/IgG3 Hinge-Fc Domains Shuffling

Human IgG3 antibodies have generally enhanced CDC compared to human IgG1antibodies, this due in part because IgG3 Fc has higher C1q-bindingaffinity than IgG1 Fc (Schumaker V N et al., Biochemistry, 1976,15:5175-81.). A shuffling strategy of the human IgG1 hinge and constantdomains with the hinge and constant domains of the human IgG3 wasundertaken to generate chimeras of anti-VLA-2 IgG4 with enhanced CDC.

Two chimeras were constructed. A first chimera based on the anti-VLA-2IgG4 was engineered to fuse the CH1 and the hinge from human IgG1 to theFc portion of human IgG3 and referred herein as heavy chain anti-VLA-2IgG-1133 (SEQ ID NO: 47); while a second construct based on anti-VLA-2IgG3 was engineered to substitute the hinge from human 19G3 with thehinge from human IgG1, this later chimera is referred herein as heavychain anti-VLA-2 IgG-3133 (SEQ ID NO: 48).

To create the anti-VLA-2 lgG-1133 heavy chain cDNA coding sequence (SEQID NO: 47), the part of heavy chain cDNA for anti-VLA-2 lgG1 encodingthe CH2 and CH3 constant domains (encoding Kabat residues 231 to itsc-terminus) in the expression vector for the anti-VLA-2 lgG1 wasreplaced with the corresponding part of a human lgG3 heavy chain gene(NCBI GenBank accession no. X03604.1, residues 161 to 377). For theanti-VLA-2 lgG-3133 heavy chain cDNA coding sequence (SEQ ID NO: 48),the part of heavy chain cDNA for anti-VLA-2 lgG3 encoding the hingeregion (ELKTPLGbTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPfrom NCBI GenBank accession no. X03604.1) (SEQ ID NO: 58) in theexpression vector for anti-VLA-2 lgG3 heavy chain was replaced with thecorresponding part (EPKSCDKTHTCPPCP) (SEQ ID NO: 59) of a human lgG1heavy chain gene (NCBI GenBank accession no. J00228.1).

Amino Acid Mutant Anti-VLA2 Antibodies with Enhanced CDC

A number of variants of the anti-VLA-2 IgG1 were designed with the goalof enhancing complement dependent cytotoxicity (CDC). In the same waythat Fc interactions with Fcγ receptors mediates ADCC, Fc interactionswith the complement component C1q mediates CDC. Although there iscurrently no 3D structure available for the Fc/C1q complex, severalstudies have mapped the binding site on human IgG for C1q to a regioncentred on residues D270, K322, P329 and P331 (Idusogie et al., JImmunol Methods 2000, 164:4178-4184). Amino acid modifications weredesigned in the D269-K334 region of the CH2 domain to explore variantsthat may mediate enhanced CDC for the anti-VLA-2 IgG1 antibody.

To create these variant cDNA coding sequences, a cDNA coding theanti-VLA-2 IgG1 heavy chain (SEQ ID NO: 44) cDNA was mutated to includethe following substitutions: S324N (referred herein as anti-VLA-2IgG1-S324N; SEQ ID NO: 49), E269D (referred herein as anti-VLA-2IgG1-E269D; SEQ ID NO:50), and S298A (referred herein as anti-VLA-2IgG1-S298A; SEQ ID NO: 51). Further variants were created by combingthese point mutations in pairs: E269D, S298A were combined to generate avariant referred herein as anti-VLA-2 IgG1-E269D/S298A (SEQ ID NO: 52);another combination referred herein as anti-VLA-2 IgG1-E269D/S324N (SEQID NO: 53) consisted of E269D and S324N; a third combination referredherein as anti-VLA-2 IgG1-S298A/S324N (SEQ ID NO: 54) combined S298A andS324N mutations. Finally all three mutations were added to create avariant referred herein as anti-VLA-2 IgG1-E269D/S298A/S324N (SEQ ID NO:55).

These variant coding DNA sequences were ligated in a vector that isbased on a modified pREP4 (Invitrogen, Calif., USA) vector carrying CMVpromoter and Bovine Growth Hormone poly-adenylation signal. In thisexpression-vector, secretion was driven by the murine VJ2C leaderpeptide.

Production of Anti-VLA2 Antibody Variants

For transient expression, equal quantities of each vector heavy chain(above) and anti-VLA-2 kappa light chain vector were co-transfected intosuspension-adapted HEK-EBNA cells (ATCC-CRL-10852) usingPolyethyleneimine (PEI). Typically, 100 ml of cells in suspension at adensity of 0.8-1.2 million cells per ml is transfected with a DNA-PEImixture containing 50 μg of expression vector encoding the variant heavychain and 50 μg expression vector light chain. When recombinantexpression vectors encoding each engineered chain genes are introducedinto the host cells, the construct is produced by further culturing thecells for a period of 4 to 5 days to allow for secretion into theculture medium (EX-CELL 293, HEK293-serum-free medium, Sigma, Buchs,Switzerland), supplemented with 0.1% pluronic acid, 4 mM glutamine, and0.25 μg/ml geneticin. The construct was then purified from cell-freesupernatant using recombinant Streamline rProtein A media (GE HealthcareEurope GmbH, Glattbrugg, Switzerland), and used for further analysis.

The expression levels of some of these variants are listed in Table 1.

TABLE 1 Transient expression levels of anti-VLA2 antibody variantsAntibody Expression (mg/L) anti-VLA-2 IgG1 30 anti-VLA-2 IgG2 8anti-VLA-2 IgG3 1 anti-VLA-2 IgG1-S324N 10 anti-VLA-2 IgG-1133 4anti-VLA-2 IgG-3133 1.4Complement Mediated Toxicity on HT1080 Cells

A cell-based assay was used to measure the capacity of the variants tomediate CDC. Lysis was measured using release of lactate dehydrogenase(LDH) to monitor lysis of variant-opsonized HT1080 cells by baby rabbitcomplement (Harlan Laboratories, C-0099F, AN VENRAY, The Netherlands).Target cells (HT1080, ATTC® No. CCL-121) were washed 2 times withcomplete medium (RPMI-1640 medium (Chemie Brunschwig AG, PAA, Basel,Switzerland) supplemented with 10% fetal bovine serum (FBS, ChemieBrunschwig AG, PAA, Basel, Switzerland) and 1% Ultraglutamine (Lonza,Verviers, Belgium)) by centrifugation and resuspension.Variant-antibodies were added at the final concentration of 1 μg/ml.Baby rabbit serum was diluted to 7.5% with complete medium and added toantibody-opsonized target cells. Plates were incubated for 3 hours at37° C. Cell cytotoxicity was measured using the Cyto Tox 96Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, USA).

FIG. 1 shows typical results for this assay with data performed intriplicate±standard deviation. In this assay, little specific lysis dueto IgG1 control (data point no. 12) or the parental anti-VLA-2 IgG4antibody (data point no. 1) or the anti-VLA-2 IgG2 (data point no. 3)antibody is observed. While no to very low level of CDC are expectedfrom the latter naturally occurring isotypes, it is rather surprisingthat the anti-VLA-2 IgG1 (data point no. 2) did not lead to anysignificant increase in CDC as it was anticipated that an IgG1 antibodyisotype had superior CDC to the IgG4 and IgG2 isotypes. Only when theS324N mutation is present, CDC is increased by at least 6.4 fold foranti-VLA-2 IgG1-S324N (data point no. 7), 6.6 fold for anti-VLA-2IgG1-S298A/S324N (data point no. 11), and 9 fold for anti-VLA-2IgG1-E269D/S298A/S324N (data point no. 10). Complement-induced lysis wasalso greatly increased for antibody variants having components of humanIgG3 antibody isotype constant region, CDC was at least increased 5.6fold for anti-VLA-2 IgG-1133 (data point no. 5), and at least increased8.6 fold for anti-VLA-2 IgG-3133 (data point no. 6). Best CDC increasewas observed for the naturally occurring variant anti-VLA-2 IgG3 (datapoint no. 4) with an enhancement of at least 9.6 fold over the parentalanti-VLA-2 IgG4 antibody. In conclusion, these results provide evidencethat the S324N mutation in the context of the anti-VLA-2 IgG1 antibodyis able to restore potent Complement-induced lysis, while the anti-VLA-2IgG3 variant is the most potent of the anti-VLA2 antibody variants ateliciting CDC.

Example 2: Humanized Anti-Alpha2 Integrin Antibody Variants withEnhanced Antibody-Dependent Cellular Cytotoxicity (ADCC)

Anti-VLA2 antibody variants investigated in the study described inExample 1 were assessed for their ability to elicit ADCC.

ADCC activities of antibodies were measured by lactate dehydrogenase(LDH)-releasing assay using the CytoTox 96 Non-Radoactive CytotoxicityAssay kit (Promega, Madison, USA). Human peripheral blood mononuclearcells (PBMC) were purified from citrated whole blood by standardFicoll-paque separation, resuspended in complete medium (RPMI-1640medium (Chemie Brunschwig AG, PAA, Basel, Switzerland) supplemented with10% fetal bovine serum (FBS, Chemie Brunschwig AG, PAA, Basel,Switzerland), 2 mM ultraglutamine 1 (Lonza, Verviers, Belgium) and 1%penicillin/streptomycin (Chemie Brunschwig AG, PAA, Basel,Switzerland)), and 100 U/ml of human IL-2 (Sigma, Mo., USA)) andincubated overnight at 37° C. The following day, PBMC were collected bycentrifugation, washed twice and resuspended in culture medium at adensity of 8×10⁶ cells/ml. The cell line HT1080 was used as targetcells. HT1080 cells were washed twice and resuspended in complete mediumat a density of 0.2×10⁶ cells/ml. Fifty microliters of antibody dilutedat 1.5 μg/ml, with a final concentration of 0.1 μg/ml (FIG. 2) or 0.01μg/ml (FIG. 3) were mixed with 50 μl of target cells, and added to anequivalent volume of PBMC into a U-bottomed 96-well plate. A target toeffector ratio of 1:40 was used throughout the experiments. After 4hours incubation at 37° C., cells were centrifuged and 50 μl samples ofcell-free supernatant were collected, transferred to a flat-bottomed96-well plate, and assayed. Percentage of lysis was calculated asfollows: (Sample release−Target spontaneous release−Effector spontaneousrelease)/(Maximum release−Target spontaneous release)*100; where Targetspontaneous release is the fluorescence from wells which only containedtarget cells, Effector spontaneous release is the fluorescence fromwells which only contained effector cells, and Maximum release is thefluorescence from wells containing target cells which have been treatedwith lysis buffer. Background percentage of lysis obtained in absence ofantibody (Target+Effector cells) was subtracted from the percentage oflysis of sample; data shown are the mean cytotoxicitypercentage±standard deviation of triplicate wells using PBMC isolatedfrom one donor.

FIG. 2 show little specific ADCC due to IgG1 control antibody or theparental anti-VLA-2 IgG4 antibody (data point no. 1 and 12,respectively); data shown demonstrate that naturally occurring humanIgG1 isotype of anti-VLA2 antibody has enhanced cellular cytotoxicitytowards VLA-2⁺ expressing cells, and more preferably anti-VLA2-IgG1antibody point mutants with the following rank order of potency:anti-VLA2-IgG1-S298A/S324N (data point no. 11; 2 foldincrease)>anti-VLA2-IgG1-S298A (data point no. 8; 1.8 foldincrease)>anti-VLA2-IgG1 (data point no. 2; 1.7 fold increase) oranti-VLA2-IgG1-E269D/S298A/S324N (data point no. 10; 1.7 foldincrease)>anti-VLA2-IgG1-E269D (data point no. 9; 1.5 fold increase).

FIG. 3 show that a similar rank order of ADCC potency for the anti-VLA2antibody variants is maintained at a ten-fold dilution of antibody.

The invention claimed is:
 1. An antibody comprising a variant human IgGFc region comprising amino acid substitution S298A that replaces serineat amino acid position 298 of a parent antibody with alanine, and aminoacid substitution S324N replacing serine at amino acid position 324 ofthe parent antibody with asparagine, wherein the numbering of the aminoacid positions is that of the EU index as in Kabat, wherein parentantibody is an antibody with a native human IgG1 heavy chain constantregion, wherein variant IgG Fc region is identical to the native humanIgG1 heavy chain constant region except for the S298A and S324N aminoacid substitutions, and wherein the antibody exhibits improved CDC andADCC as compared to the parent antibody.
 2. The antibody of claim 1,wherein the variant C_(H) comprises the amino acid sequence set forth asSEQ ID NO:
 42. 3. A composition comprising the antibody of claim 1 and apharmaceutically acceptable carrier.
 4. A kit comprising the antibody ofclaim 1 and optional instructions.
 5. The antibody of claim 1, whereinthe antibody is selected from the group consisting of a chimericantibody, a humanized antibody and a fully human antibody.
 6. Theantibody of claim 1, wherein the antibody is a humanized antibody. 7.The antibody of claim 1, wherein the antibody is a fully human antibody.8. The antibody of claim 1, wherein the antibody is a anti-α2 integrinantibody.
 9. The antibody of claim 8, comprising a light chain variableregion sequence of any one of SEQ ID NOs:22-33, and a heavy chainvariable region sequence of any one of SEQ ID NOs:8-19.
 10. The antibodyof claim 8, comprising a light chain variable region and a heavy chainvariable region wherein the light chain variable region comprises: anLCDR1 comprising the amino acid sequence SAQSSVNYIH (SEQ ID NO:4); anLCDR2 comprising the amino acid sequence DTSKLAS (SEQ ID NO:5); and anLCDR3 comprising the amino acid sequence QQWTTNPLT (SEQ ID NO:6), andwherein the heavy chain variable region comprises: an HCDR1 comprisingthe amino acid sequence GFSLTNYGIH (SEQ ID NO:1); an HCDR2 comprisingthe amino acid sequence VIWARGFTNYNSALMS (SEQ ID NO:2); and an HCDR3comprising the amino acid sequence ANDGVYYAMDY (SEQ ID NO:3).